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Induction of hypersignaling as a therapeutic approach for treatment of BCR-ABL1 positive Acute Lymphoblastic Leukemia (ALL) cells
by
Seyedmehdi Shojaee
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
May 2013
Copyright 2013 Seyedmehdi Shojaee

Current therapy approaches for tyrosine kinase driven leukemia including Ph⁺ ALL and CML are almost entirely focused on the development of more potent tyrosine kinase inhibitors (TKI). The ultimate goal in this approach is to reduce the oncogenic signaling below a minimum threshold that is required for the survival of leukemia cells. Despite the successful results obtained so far, this approach has its own drawbacks. Unfortunately, TKI treatment is not very effective in Ph⁺ ALL patients. Furthermore, in CML patients it can lead to the appearance of resistant tumors that are not sensitive to therapy anymore. Therefore, new strategies for killing the Ph⁺ leukemia independent of kinase inhibition are necessary. ❧ Hypersignaling has been known as an unfavorable condition for the survival of cells, and induction of hypersignaling may result in stalled proliferation or cellular senescence. However, its implication as a therapeutic mean has not been well-characterized. One strategy for induction of hypersignaling is to suppress the inhibitory regulators of signaling pathways. Therefore, we hypothesized that inhibition of negative feedback regulators or inhibitory phosphatases in Ph⁺ kinase leukemia could raise the signaling level and induce cell cycle arrest or senescence in transformed cells. Importantly, cells lacking high oncogenic kinase activity should be insensitive to such inhibition of negative feedback signaling. ❧ In chapter two of this thesis, we focused on the mitogen-activated protein kinase (MAPK) pathway. By the analysis of gene expression changes and evaluation of protein levels, we found that DUSP6 and SPRY2, two negative feedback regulators of the MAPK pathway, are highly expressed in human B cell lineage Ph⁺ ALL, while they are not expressed in B cell progenitor cells. Interestingly, in contrast to BCR-ABL1 kinase driven leukemia, non kinase-driven B cell Non-Hodgkin-Lymphoma (B-NHL) lack expression of these genes. To study the function of DUSP6 and SPRY2 in a genetic mouse model of Ph⁺ like leukemia, we transduced bone marrow pre-B cells from DUSP6-/-, SPRY2fl/fl mice and respective wildtype controls with retroviral BCR-ABL1. Defects in either of these two negative feedback mediators caused profound signaling imbalances in BCR-ABL1 leukemia cells and resulted in drastic negative consequences for the cells. For instance, Dusp6-deficient leukemia cells rapidly underwent cellular senescence and were less capable of colony formation in semisolid culture. In addition, Lack of Dusp6 and Spry2 dramatically increased cellular reactive oxygen species (ROS). Furthermore, inducible Cre-mediated deletion of Spry2 in leukemia cells resulted in rapid cell death. ❧ To test whether negative feedback signaling molecules represent a potential target for pharmacological inhibition in the treatment of kinase driven leukemia, we tested the DUSP6 small molecule inhibitor 2-benzylidene-3-(cyclohexylamino)-1-Indanone hydrochloride (BCI). BCI induced massive accumulation of ROS and subsequent cell death in a panel of patient-derived cases of Ph⁺ ALL, including two cases with T315I mutation. In vivo and ex vivo studies showed that BCI can increase the survival of NOD/SCID recipient mice transplanted with patient-derived Ph⁺ ALL cells with T315I mutation. ❧ In chapter three, we turn to the inhibitory phosphatases in Ph⁺ ALL cells. We found that three key inhibitory phosphatases (INPP5D/SHIP1, PTEN and PTPN6/SHP1) are expressed at high levels in these cells. INPP5D and PTEN negatively regulate AKT mediated signaling by dephosphorylating phosphatidylinositol-3,4,5-trisphosphate, at 5- and 3-phosphates, respectively. In addition, PTPN6 and INPP5D can both be recruited to the ITIM motifs of various inhibitory surface receptors and negatively regulate activation signals from tyrosine kinases and activating receptors. ❧ We showed that Cre-mediated deletion of Pten, Inpp5d and Ptpn6 in B cell lineage BCR-ABL1 ALL cells resulted in increased signaling downstream of the BCR-ABL1 kinase. Interestingly, this hypersignaling resulted in a variety of negative outcomes for the ALL cells such as drastic upregulation of reactive oxygen species (ROS), accumulation of DNA damage molecules, increased levels of cellular senescence and cell death. ❧ Studying B cell lineage BCR-ABL1 transformed Inpp5dfl/fl, Ptenfl/fl and Ptpn6fl/fl leukemia cells in vivo, we observed that deletion of these inhibitory phosphatases resulted in prolonged survival of leukemia transplant recipient mice. In addition, using small molecule inhibitors of INPP5D (3AC) and PTEN (VO-OHpic) recapitulated the genetic findings and killed patient-derived leukemia cells carrying the T315I mutant BCR-ABL1. ❧ We also found that one of the side effects of hypersignaling is the crosstalk between different signaling pathways. Surprisingly, induction of hypersignaling in the MAPK or PI3K/AKT pathways caused downregulation of the JAK/STAT pathway. While STAT5 activity is necessary for survival of the Ph+ leukemia cells, hypersignaling in other two pathways lead to acute dephosphorylation of STAT5. ❧ In summary, our studies identify negative feedback regulators of the MAPK pathway (including DUSP6 and SPRY2) and inhibitory phosphatases such as PTEN, INPP5D and PTPN6 as novel therapeutic targets in BCR-ABL1 positive B cell lineage leukemia. Pharmacological blockade of these molecules represents a fundamentally novel and powerful approach to increase oncogenic signaling beyond a tolerable threshold, thus causing excessive accumulation of ROS, crosstalk between different signaling pathways and subsequent cell death.

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i
Induction of hypersignaling as a therapeutic approach for treatment of BCR-ABL1 positive Acute Lymphoblastic Leukemia (ALL) cells
by
Seyedmehdi Shojaee
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
May 2013
Copyright 2013 Seyedmehdi Shojaee